Affinity purification ("affinity chromatography") broadly refers to separation methods based on a relatively high binding capacity ("affinity") of a target material to be purified, generally termed a "ligate", for a complementary ligand. While such purifications can be accomplished in solution, more typically, a ligand selected inter alia for selectivity toward the target material is immobilized on a supporting matrix; the mixture to be purified with respect to the ligate is then exposed to the matrix-bound ligand, and the resulting ligate/ligand complex is subsequently dissociated and the ligate recovered, if desired. The procedure has had particular application in the separation of biological materials, especially biological macromolecules such as proteins.
While biological purifications based on such affinity separations are relatively straightforward in theory, numerous problems have arisen in practice, particularly with respect to obtaining matrix systems adapted for rapid separation and optimum yield and purity of the target material. Ideally, matrices for ligand immobilization should have a large surface area and comprise an open and loose porous network to maximize interaction of matrix-bound ligand with ligate during the separation procedure. The matrix should be chemically and biologically inert, at the very least toward the ligand and ligate; be adapted for ligand immobilization; and be stable under reaction conditions employed, for example during matrix activation, ligand binding, and ligand-ligate complex formation, especially with respect to the solvent, pH, salt, and temperature employed. The matrix should also be stable for a reasonable length of time under ordinary storage conditions. To minimize competition for the target material and maximize purity of recovered product, supports for immobilization of ligands, especially biospecific ligands, should be free from extraneous ion exchange sites, and should not promote non-specific bonding. Matrices, especially those used in pressurized affinity separation techniques, should be mechanically strong and be able to withstand at least the moderate pressures typical of these conventional systems (up to about 5 bar, for example). Further, since matrices are frequently derivatized, for example to promote ligand immobilization or to permit improved ligand/target interaction, the matrix should be readily derivatizable to these ends, preferably at room temperature and in aqueous media, without the use of toxic chemicals; the derivatized matrix should also meet the above criteria.
A number of useful matrix materials have been identified over the years since the principles of affinity chromatography were first enunciated, including agarose gels; cellulose; dextran; polyacrylamide; hydroxyalkylmethacrylate gels; polyacrylamide/agarose gels; ethylene copolymers, especially with polyvinyl acetate; copolymers of methacrylamide, methylene bis-methacrylamide, glycidyl-methacrylate and/or allyl-glycidyl-ether (such as Eupergit C, Rohm Pharma, Darmstadt, West Germany); and diol-bonded silica.
These and similar known matrix materials have been in the past typically coupled with the selected ligand and distributed in chromatographic columns in bead form. The material to be purified is then passed through the column; the target material is adsorbed by the beads, and, if desired, subsequently recovered by desorption of the ligate and elution from the column. Beads in current use commonly range in size from about 60 to about 300 mesh (U.S. standard), and accordingly must have adequate porosity to permit access of the target material to internally-bound ligands for efficient separation; preparation of beads of both good porosity and stability has proved difficult. Further, since chromatographic bead columns are susceptible to clogging with biological debris leading to rise in pressure and collapse of the beads, particulates must be pre-filtered from the material to be purified before affinity separation is undertaken; this often causes product loss, and in many instances (e.g., wherein the ligate is derived from a genetically-engineered microorganism), adequate removal of particulates from the crude preparation is difficult or impossible.
To overcome these problems, techniques have recently been developed employing microporous membranes or films rather than beads as the preferred matrix form for immobilizing ligands for affinity separations. In affinity separation systems employing microporous membranes, the crude filtration fluid containing the ligate flows through the membrane, typically under pressure, by convection processes which rapidly bring the ligate immediately into the vicinity of the affinity ligands immobilized on the membrane, in contrast to the much slower diffusion processes which characterize the crude fluid flow over matrices typical of the conventional bead chromotography separations described above. Additionally, owing to the large surface area of these membranes, the attached ligands have a high exposure to the ligates present in the material to be purified.